PROJECT DESCRIPTION Human pancreatic ribonuclease (RNase 1) is a secretory enzyme that can block the flow of biochemical information by catalyzing the cleavage of cellular RNA. Our intent is to use ideas and methods from biological chemistry, molecular biology, and cell biology to reveal key mechanistic aspects of the cytotoxicity of human RNase 1 in physiological and (potentially) clinical settings. During the next grant period, this intent will be achieved in four Specific Aims. Specific Aims. In Aim 1, we will determine whether the complex of RNase 1 with the cytosolic ribonuclease inhibitor protein (which has femtomolar affinity for RNase 1) acts as a ?sensor? for oxidative damage within a human cell. In Aim 2, we will determine the structural and physiological roles of the N-linked glycans that are installed on RNase 1. In Aim 3, we will develop an RNase 1 zymogen as a ?prodrug? that is activated by matrix metalloproteases. In Aim 4, we will evaluate RNase 1 as an endogenous antimicrobial agent. Significance. The results of the research proposed herein will provide a detailed biochemical understanding of the cytotoxic activity of RNase 1, and could ultimately lead to new chemotherapeutic agents based on an endogenous human enzyme.